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Epitope based on IL-6 and application of epitope

An antigen epitope, IL-6 technology, applied in the field of biomedicine, can solve the problems of high price and long service life of antibody drugs

Active Publication Date: 2015-12-30
浓孚雨医药河北有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antibody drugs have a long use cycle, are expensive, and are prone to immune tolerance. Active immunotherapy strategies based on IL-6 vaccine are becoming a new hot spot in RA immunotherapy research.

Method used

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  • Epitope based on IL-6 and application of epitope
  • Epitope based on IL-6 and application of epitope
  • Epitope based on IL-6 and application of epitope

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] 1. Transplantation of linear B epitopes

[0157] In this example, 6 fusion epitopes were designed, and DTT was used as the carrier to prepare the basis for immune antigen peptides. The transplantation results are summarized in Table 1.

[0158] Table 1 The design of IL-6 epitope transplantation to DTT

[0159]

[0160] 2. Preparation of Antigenic Peptides

[0161] 2.1 Primer design

[0162] According to the human IL-6 domain, murine IL-6 domain, five candidate epitopes designed and the base sequence information of DTT, primers were designed using Primer5.0 software. Among them, the two structural domains are respectively inserted at the carboxy terminus (C terminus) of DTT, and the designed primers are named hP1, hP2, mP1, mP2. The insertion of the epitope into DTT requires the design of four primers, named P1, P2, P3, and P4, respectively, wherein the primers P1, P4 are consistent, and BamHI is inserted at the 5' end and 3' end of the P1, P4 primers respectively ...

Embodiment 2

[0209] Embodiment 2 animal immunity and activity detection

[0210] 1 Animal immunity and determination of antigen-antibody binding strength

[0211] 1.1 Immunization and blood collection of animals

[0212] Sixty BalB / c mice aged 6-8 weeks were randomly divided into 11 groups, aluminum adjuvant control group, empty vector DTT control group, and 8 experimental groups, 6 mice in each group. The antigen was mixed with aluminum hydroxide (final concentration 1.6 mg / mL) adjuvant 1:1, and the injection dose was 50 μg antigen / animal / time, and multi-point subcutaneous injection on the back. Immunization was performed every two weeks after the first immunization, and antiserum was collected one week after the third immunization.

[0213] 1.2 ELISA method to detect the production of antibodies

[0214] In order to determine whether antibodies against hIL6 or mIL6 are produced in the serum of the experimental group, an ELISA experiment is required. The process of ELISA assay antibody ...

Embodiment 3

[0237] Example 3 Determination of the activity of neutralizing interleukin-6 by multiple antibodies

[0238] 1. Purification and quantification of polyclonal antibodies

[0239] The polyclonal antibody was purified by conventional methods in the field, and the purity of the polyclonal antibody obtained by electrophoresis analysis was over 90%, and the polyclonal antibody was quantified by using the BCA method.

[0240] 2. Exploration of the dosage of multiple antibodies to neutralize IL-6 activity in vitro

[0241] 1) Take out 1 tube (about 107 cells / mL) of 7TD1 cells frozen in liquid nitrogen (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) in an ice box, freeze and thaw quickly at 37°C, add 7 mL of RPMI1640 medium and mix well Centrifuge to remove supernatant (wash away DMSO immediately);

[0242] 2) Resuspend 500 μL of cell cryopreservation medium in 7 mL RPMI1640 medium (containing 10% FBS, 128 pg / mLmIL6) and 7 mL RPMI1640 medium (containing 10%...

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Abstract

The invention discloses an epitope based on IL-6 and application of the epitope and particularly provides epitope peptide. The epitope peptide is derived from mammal IL-6 and includes one or more epitopes. The length of the amino acid sequence of the epitope is 2-100% the whole length of the IL-6. The length of the epitope peptide is 3-300 amino acids. Besides, recombinant protein composed of the epitope peptide and carrier protein can induce immune response, to the IL-6, generated by mammals of the same time. The invention further provides fusion protein containing the epitope peptide. The recombinant protein prepared through fusion expression of the epitope peptide and the carrier protein is used and can effectively activate the immune response, to the IL-6, of an organism.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to an IL-6-based epitope and its application. Background technique [0002] Rheumatoid arthritis (RheumatoidArthritis) is one of the most common autoimmune diseases. The disease is a long-term chronic inflammation of the joints caused by the body's immune system's abnormal attack on the joints of the human body. Drug research for the treatment of rheumatoid arthritis (RA) has been conducted for many years. The previous strategy was to take anti-inflammatory drugs first, and once the symptoms of joint damage were detected clinically, control rheumatoid drugs would be started. Drugs for the control of rheumatoid diseases are generally divided into two categories: heterologous biological agents and bioengineered agents. Xenobiotic agents are a class of molecular compounds that cannot be produced or synthesized by the body in a natural state, such as common cyclospor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/54C07K19/00C12N15/12C12N15/62C12N15/63A61K38/20A61K48/00A61K39/00A61P37/00A61P35/00A61P9/10A61P9/00
Inventor 李荣秀都伟杰
Owner 浓孚雨医药河北有限公司
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